BMP-2-Loaded Self-Crosslinking CaP/Hydrogel Composite Enables Complete Regeneration of Critical-Sized Segmental Bone Defects

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study evaluates the combination of a CaP/hydrogel composite that delivers BMP-2. A more scientifically correct grouping would be: (1) a control scaffold delivering BMP-2, e.g. collagen sponge; and (2) the CaP/hydrogel composite delivering the same dose of BMP-2. The current grouping does not prove that CaP/hydrogel can deliver BMP-2, it just proves that the BMP-2 is working. Bone overgrowth (ectopic bone formation) seems to be significant in the BMP-2 group, this usually mean the BMP-2 is overdosed and is not control-released from the scaffold.

The authors could improve this paper by adding at least in vitro data showing the release profile of BMP-2 from the composite.

Other comments: 

Method:

- More details of BMP-2 reconstitution buffer.

- More details of micro-CT sample preparation: was the plate firmly attached or loosen after 20 weeks? Did the bone attach to the plate? How the plate was removed?

- SEM & EDX: how many sections were analysed for each animal? Does it include the top/mid/bottom layer of the defect?

Result:

Micro-CT analysis:

- Regarding the stability of the implants after 20 weeks, it is more convincing to show all images as done in Figure 2.

- “The regenerated tissue exhibited a three-dimensional architecture similar to native bone” – the structure does not look like native bone at this view. The authors could show cross-section of the 3D reconstructed images, with more close-up view to show the architecture more clearly.

- “Residual ceramic volume was markedly reduced in this group, suggesting ac-tive biomaterial remodeling.” – The authors should do quantitative analysis as done in the 2D (figure 2), e.g. BV/TV of new bone, scaffold volume/TV within ROI to support this result.

- - Figure 1: please add the scale bar.

SEM & EDX analysis:

- The authors should indicate this analysis was based on how many histology sections.

Histology analysis:

- I can’t see A-B-C-D; the labels (bone, BCP granule, soft tissue) are poorly colored and placed; scale-bar is too small.

Author Response

The study evaluates the combination of a CaP/hydrogel composite that delivers BMP-2. A more scientifically correct grouping would be: (1) a control scaffold delivering BMP-2, e.g. collagen sponge; and (2) the CaP/hydrogel composite delivering the same dose of BMP-2. The current grouping does not prove that CaP/hydrogel can deliver BMP-2, it just proves that the BMP-2 is working.

We thank the reviewer for this important comment. We agree that inclusion of a reference BMP-2 carrier such as a collagen sponge would allow a direct comparison of release systems. However, the primary objective of the present study was not to compare different BMP-2 carriers, but rather to evaluate whether our self-crosslinking CaP/Si-HPMC composite could support bone regeneration and biomaterial remodeling in a clinically relevant large-animal segmental defect model.

Our experimental design was based on comparing the scaffold alone versus the scaffold loaded with rhBMP-2 in order to isolate the contribution of BMP-2 within this composite system. Therefore, we acknowledge that the current study demonstrates the efficacy of rhBMP-2 delivered from SCBS, rather than superiority over other delivery systems.

We have added this limitation to the Discussion section and now explicitly state that future studies should compare SCBS with clinically established carriers such as collagen sponges and include direct BMP-2 release kinetics analysis.

Bone overgrowth (ectopic bone formation) seems to be significant in the BMP-2 group, this usually mean the BMP-2 is overdosed and is not control-released from the scaffold.

We thank the reviewer for this relevant observation regarding ectopic bone formation. We agree that excessive BMP-2 dose and uncontrolled release can lead to ectopic ossification in some delivery systems. However, in our model, we believe that the limited ectopic bone observed is more likely related to mechanical displacement of the implanted composite rather than uncontrolled diffusion of BMP-2 alone.

Indeed, the CaP/Si-HPMC composite is implanted as a viscous and moldable paste. After in situ crosslinking, the hydrogel forms a three-dimensional network that stabilizes the construct but remains relatively compliant and not fully rigid compared with preformed solid scaffolds or hardened cements. Consequently, under postoperative mechanical loading and local micromotions, small portions of the composite may have shifted outside the initial defect boundaries.

Importantly, SEM observations support this interpretation, as the ectopic bone areas also contained residual BCP granules. This suggests that the ectopic mineralized tissue formed around displaced composite fragments containing both BMP-2 and BCP, rather than resulting from BMP-2 diffusion alone. We have clarified this point in the revised Discussion section:

“A limited amount of ectopic bone formation was observed in some samples. This may not solely reflect uncontrolled BMP-2 diffusion but could also result from slight displacement of the CaP/Si-HPMC composite after implantation. Because the hydrogel remains relatively compliant after crosslinking, postoperative micromotions may induce migration of composite fragments containing both BMP-2 and BCP granules, thereby promoting localized bone formation outside the initial defect boundaries.”

 

 

The authors could improve this paper by adding at least in vitro data showing the release profile of BMP-2 from the composite.

We agree that in vitro BMP-2 release kinetics would further strengthen the mechanistic interpretation of this study. However, such data are not available in the current work. We acknowledge this limitation and have added it to the Discussion as an important direction for future investigation. At least we performed ELISA évaluation of BMP2 of the supernatant after day 3 of SCBS in different solvents at 1/10 and 1/500 dilutions. BMP2 ELISA assay at day 3 at 1/10 and 1/500 dilutions in different solvents showed that almost no rapid release was observed. (S2)

 

 

Other comments: 

Method:

- More details of BMP-2 reconstitution buffer.

We thank for the remark, we added in the text “using the liquid Pfizer kit”

- More details of micro-CT sample preparation: was the plate firmly attached or loosen after 20 weeks? Did the bone attach to the plate? How the plate was removed?

We thank for the remark, The osteosynthesis plate (eight-hole LCP® 2.4 mm Depuy-Synthes (Locking Compression Plate)) was always attached after 20 weeks.

We add in the text modifications in MAT&MET and in the discussion

- SEM & EDX: how many sections were analysed for each animal? Does it include the top/mid/bottom layer of the defect?

We thank for the remarks and questions and added in the MAT&MET  section : For each animal model (8-year-old female beagle), four (4) samples were analyzed by SEM and EDX, two with BMP (left ulna) and two without BMP (right ulna).

The explants were inserted in a medio-distal (cranio-caudal) direction ; each block was sectioned longitudinally in the middle (yielding two samples). This included the upper, middle, and lower layers.

Microanalysis was performed using energy-dispersive X-ray spectroscopy coupled to scanning electron microscopy with an accelerating voltage of 100 kV.

The calcium/phosphorus (Ca/P) ratio was compared in the newly formed bone observed in samples treated or not with BMP2 after explantation, as well as with the Ca/P ratio of the adjacent native bone. The Ca/P ratio is expressed as mean ± standard deviation obtained from 16 measurements on each sample.

For histological analysis, five slides were analyzed for each sample.

 

Result:

Micro-CT analysis:

- Regarding the stability of the implants after 20 weeks, it is more convincing to show all images as done in Figure 2.

We thank the reviewer for this valuable suggestion. We agree that presenting images from all animals provides a more comprehensive illustration of implant stability and the reproducibility of the results. Therefore, we have included the corresponding micro-CT reconstructions for all animals (Fig 1) These additional images confirm the consistent behavior of the implants and the reproducibility of the bone regeneration observed in both experimental groups. A deep learning analyze of the 3D Micro-CT reconstructions was added in M&M and result sections using Dragonfly software to confirm in 3D the results showed in 2D with the SEM image analysis.

- “The regenerated tissue exhibited a three-dimensional architecture similar to native bone” – the structure does not look like native bone at this view. The authors could show cross-section of the 3D reconstructed images, with more close-up view to show the architecture more clearly.

We thank the reviewer for this helpful suggestion. We agree that the original three-dimensional reconstruction does not fully illustrate the internal architecture of the regenerated tissue. To better support our statement, we have added cross-sectional views and higher-magnification images of the micro-CT reconstructions in the revised Figure 1. These additional views more clearly demonstrate the continuity of the regenerated bone, its trabecular organization, and its integration with the host bone, thereby providing a more accurate visualization of the bone architecture.

- “Residual ceramic volume was markedly reduced in this group, suggesting active biomaterial remodeling.” – The authors should do quantitative analysis as done in the 2D (figure 2), e.g. BV/TV of new bone, scaffold volume/TV within ROI to support this result.

We thank the reviewer for this valuable suggestion. We agree that quantitative three-dimensional micro-CT analysis would provide additional information regarding bone formation and residual biomaterial volume. However, in the present study, micro-CT was primarily intended to provide a qualitative assessment of defect bridging and the spatial distribution of mineralized tissues. Quantitative evaluation of newly formed bone and residual BCP was performed by SEM-based histomorphometry, which enabled reliable discrimination between bone, biomaterial, and soft tissue at high resolution. These quantitative data are presented in Figure 3 and demonstrate a significant increase in bone formation together with a marked reduction in residual BCP in the rhBMP-2 group. We have clarified this point in the revised manuscript and now explicitly indicate that the micro-CT observations are supported by the quantitative histomorphometric analysis.

We change the text : "Micro-CT qualitatively showed a marked reduction in the amount of residual ceramic in the rhBMP-2 group. This observation was confirmed by quantitative SEM histomorphometry (Figure 3), which demonstrated a significantly lower residual BCP fraction, supporting active biomaterial remodeling.

- - Figure 1: please add the scale bar. (Done)

SEM & EDX analysis:

- The authors should indicate this analysis was based on how many histology sections.

For each animal model (8-year-old female beagle), four (4) samples were analyzed by SEM and EDX, two with BMP (left ulna) and two without BMP (right ulna).

The explants were inserted in a medio-distal (cranio-caudal) direction ; each block was sectioned longitudinally in the middle (yielding two samples). This included the upper, middle, and lower layers.

Histology analysis:

- I can’t see A-B-C-D; the labels (bone, BCP granule, soft tissue) are poorly colored and placed; scale-bar is too small.

We thank the reviewer for this remark. We changed the labeling of the picture and add the size of the scale bars.

Reviewer 2 Report

Comments and Suggestions for Authors

This laboratory study evaluates the use of a composite based on biphasic calcium phosphate supplemented with hydroxypropyl methylcellulose and recombinant human BMP-2. The evaluation performed in an animal model is highly valuable to demonstrate the safety and efficacy of its use in clinical practice, especially since a critical-size defect model with significant scientific relevance was proposed. For this reason, I believe this manuscript has potential for publication. However, I suggest that some points should be revised:

 

Introduction

- This section is very well written and provides a solid background on the topic.

 

Materials and Methods

- Some abbreviations, such as SCBS, were already explained in the Introduction; however, these terms are redundantly defined again throughout the Methods section. Please adjust accordingly.

- It was stated that a modified protocol was used for the reconstitution of rhBMP-2 to be incorporated into the hydrogel. Please specify the protocol used.

- Was a sample size calculation performed to determine the number of animals included in the study? Please indicate the calculation performed or justify why it was not conducted.

- Please indicate the age of the animals in weeks, as well as their sex.

 

Results

- The EDX data are not presented in the Results section. I suggest including the graphs related to this analysis.

- Figure 1 presents data from only a single animal. I suggest including images from all animals.

 

Discussion

- Please indicate the limitations of the study, as well as future research perspectives based on the findings obtained.

 

Conclusion

- The conclusion is focused on the study objective.

Author Response

This laboratory study evaluates the use of a composite based on biphasic calcium phosphate supplemented with hydroxypropyl methylcellulose and recombinant human BMP-2. The evaluation performed in an animal model is highly valuable to demonstrate the safety and efficacy of its use in clinical practice, especially since a critical-size defect model with significant scientific relevance was proposed. For this reason, I believe this manuscript has potential for publication. However, I suggest that some points should be revised:

 

Introduction

- This section is very well written and provides a solid background on the topic.

 We thank the reviewer for the positive assessment of the Introduction section and for recognizing the relevance of the scientific background provided.

Materials and Methods

- Some abbreviations, such as SCBS, were already explained in the Introduction; however, these terms are redundantly defined again throughout the Methods section. Please adjust accordingly.

We thank the reviewer for this observation. Redundant definitions of abbreviations such as SCBS in the Materials and Methods section have been removed for improved readability.

- It was stated that a modified protocol was used for the reconstitution of rhBMP-2 to be incorporated into the hydrogel. Please specify the protocol used.

We thank the reviewer for this comment. Additional details regarding the rhBMP-2 reconstitution protocol, including the composition of the reconstitution buffer and the modifications introduced relative to the manufacturer’s instructions, have been added to the revised Materials and Methods section. We added in the text : “using the liquid Pfizer kit”

- Was a sample size calculation performed to determine the number of animals included in the study? Please indicate the calculation performed or justify why it was not conducted.

We thank the reviewer for this important comment. No formal a priori sample size calculation was performed. The number of animals was determined based on ethical considerations related to large-animal experimentation and according to previous studies using similar canine critical-size defect models. In addition, the bilateral design of the study, where each animal served as its own control, reduced inter-animal variability and increased the statistical power of comparisons while minimizing the number of animals required.

- Please indicate the age of the animals in weeks, as well as their sex.

We thank the reviewer for this observation. The age and sex of the animals have now been added to the revised Materials and Methods section.

Five healthy adult female beagle dogs 8 years old were included in the study.

 

 Results

- The EDX data are not presented in the Results section. I suggest including the graphs related to this analysis.

We thank the reviewer for this observation. We added a figure in the supplementary data

- Figure 1 presents data from only a single animal. I suggest including images from all animals.

 We thank the reviewer for this suggestion. Figure 1 has been changed with all the results in 3D and 2 D using micro CT.

Discussion

- Please indicate the limitations of the study, as well as future research perspectives based on the findings obtained.

This study has several limitations. First, the sample size was limited, although this is inherent to large-animal studies and partially compensated by the paired experimental design. Second, no comparison with a standard BMP-2 carrier such as a collagen sponge was performed. Third, in vitro BMP-2 release kinetics were not assessed, limiting mechanistic interpretation of growth factor delivery. Future studies should compare this composite with clinically established carriers, investigate release kinetics and dose-response relationships, and further optimize scaffold retention and mechanical stability.

 

Conclusion

- The conclusion is focused on the study objective.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no further comment.